Thrombin is a serine protease which is widely used in clinical applications in several commercial products. It is a common component of surgical dressings, and has been used in combination with fibrinogen and other proteins in hemostatic systems such as fibrin glues, adhesives, and sealants. Fibrin sealants typically comprise a fibrinogen component and a thrombin component. When both components are mixed (e.g. when applied to a bleeding wound or surgical incision) thrombin cleaves the fibrinogenpeptides off the fibrinogen thus allowing the latter to generate insoluble fibrin polymers/sealant.
Concentrated (e.g. more than 500 IU/mL), purified thrombin in aqueous liquid form may display a reduction in activity during prolonged storage, primarily as a result of autolysis. Assessment of thrombin degradation is thus an essential physico-chemical analytical tool for determining thrombin stability.
Mammalian α-thrombin is made up of two disulfide linked polypeptide chains A and B. The B chain is post-translationally modified (e.g. by glycosylation) and exhibits thrombin's proteolytic activity toward fibrinogen and other proteins. The α-thrombin can autolyze into β-thrombin, and γ-thrombin polypeptide derivatives, which can be partially identified by Gel electrophoresis and Western Blot.
Thrombin autolysis is a major challenge in manufacturing and storing of thrombin, especially at high concentrations. The methods known in the art for identifying thrombin degradation polypeptides (β-thrombin and γ-thrombin derivatives) are inadequate in that they either provide insufficient separation between thrombin and its degradation polypeptides, a denaturing separation and/or are labor intensive. Therefore, the quantitation is not accurate and/or possible.
Background art includes:
Boissel J P et al. “Covalent structures of beta and gamma autolytic derivatives of human alpha-thrombin”. J Biol Chem. 1984 May 10; 259(9):5691-5697; Chang J Y. “The structures and proteolytic specificities of autolysed human thrombin”. Biochem J. 1986 Dec. 15; 240(3):797-802; Karlsson G. “Analysis of human alpha-thrombin by hydrophobic interaction high-performance liquid chromatography”. Protein Expr Purif 2003 January; 27(1):171-174; European Patent No. EP 0443724; and WO 2004/103519.
Boissel et. al. describes the use of CEX-HPLC followed by RP-HPLC analysis to separate the different thrombin degradation polypeptides. Chang describes HPLC analysis of pure thrombin fractions separated by SEC chromatography and further analyzed using RP-HPLC. The above methods have the shortcoming of requiring at least two separation steps for quantification and separation of thrombin from other proteins.
Karlsson describes hydrophobic interaction chromatography (HIC) to separate thrombin degradation products.
European Patent No. EP 0443724 discloses a method for preparing a viral safe thrombin, however, the method is denaturing and shows no separation between the different thrombin degradation products or between the different α-thrombin post-translational variants.
WO 2004/103519 discloses methods for the separation of charged molecules such a proteins according to their isoelectric points (pI's) and includes the systems and buffering compositions employed for isolating charged molecules.
There remains an unmet need for analytical methods for quantifying α-thrombin or β-thrombin; and for the purification of active, intact α-thrombin or of β-thrombin from proteinatious solutions which overcome the above defects of the art.